Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation
- Instituto Gulbenkian de Ciência, Portugal
- Universidade de Lisboa, Portugal
Figures
Figure 1 with 4 supplements
Hml+Lz+ cells increase during larval development by de novo differentiation.
(A) Dorsal view of a third instar larva with hemocyte nuclei marked using HmlΔ-nuclearDsRed. The lymph gland (arrow) and sessile hemocytes along the body axis are visible, particularly in a big …
Figure 1—figure supplement 1
Example of a sessile hemocyte cluster (abdominal segment A7) in a HmlΔ-DsRed; Lz>mCD8GFP larva. It is possible to observe small HmlhighLzlow cells (arrows) and Hml−Lz+ cells (asterisks).
https://doi.org/10.7554/eLife.06166.004
Figure 1—figure supplement 2
Probability density plots for the different cell type sizes found in sessile clusters of HmlΔ-DsRed; Lz>mCD8GFP larvae (n = 8 samples).
Hml+Lz− plasmatocytes (red peak) are smaller than Hml+Lz− (green peak) and Hml+Lz+ (yellow peak) crystal cells.
Figure 1—figure supplement 3
Throughout the 3-hr period covered in our videos, we can observe that GFP intensity in Lz+ cells increases, as measured by mean grey value of the cell at the beginning (0 min) and at the end (180 min) of the video.
This suggests that, during crystal cell maturation, GFP driven by Lz-GAL4 increases.
Figure 1—figure supplement 4
Quantification of GFP intensity and cell area of Lz+ cells in hemolymph smears of HmlΔ-DsRed; Lz>mCD8GFP larvae, shows a strong positive correlation between cell size and GFP intensity.
https://doi.org/10.7554/eLife.06166.007
Figure 2
Lz+ cells derive from mature plasmatocytes.
(A) P1 immunofluorescence (IF) staining of sessile hemocytes marks the majority of Lz− and Lz+ cells. Bars represent the mean ratio of P1+ and P1− cells in these two population of cells (n = 10 …
Figure 3 with 5 supplements
Serrate downregulation in plasmatocytes leads to a reduction in sessile crystal cell number.
(A) Notch RNAi driven in all hemocytes reduces the number of melanized sessile crystal cells observed upon heat shock treatment to the whole larva. A similar level of reduction is seen with SerrateRN…
Figure 3—figure supplement 1
NotchRNAi and SerrateRNAi but not DeltaRNAi driven in all hemocytes reduce the number of melanized sessile crystal cells observed upon heat shock treatment to the whole larva (males are shown, n = 20).
Error bars represent the SEM. n.s. = p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3—figure supplement 2
The localization of hemocyte in sessile clusters is not affected by Notch pathway manipulation through RNAi induction under HmlΔ-GAL4 control.
The large sessile hemocyte cluster in the dorsal side of the abdominal segment A7 is highlighted in all larvae (dotted yellow circle).
Figure 3—figure supplement 3
Notch pathway manipulation through RNAi induction under HmlΔ-GAL4 control, does not change hemocyte concentration in hemolymph.
Bars represent the mean value of total hemocyte concentrations (n = 20 samples), error bars represent the SEM. n.s. = p ≥ 0.05.
Figure 3—figure supplement 4
Notch knockdown through RNAi under HmlΔ-GAL4 control does not increase cell death as measured by a flow cytometry Propidium Iodide (PI) exclusion assay , error bars represent the SEM. n.s. = p ≥ 0.05.
https://doi.org/10.7554/eLife.06166.014
Figure 3—figure supplement 5
Serrate RNAi driven only in Lz+ cells does not reduce the number of melanized sessile crystal cells seen upon heat shock treatment contrarily to two other drivers expressed in plasmatocytes, Eater-GAL4 and Pxn-GAL4 (males are shown, n = 20 samples).
Error bars represent the SEM. n.s. = p ≥ 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Figure 4 with 3 supplements
Cluster structure is necessary for crystal cell development.
(A) Sessile hemocytes in Lz>mCD8GFP HmlΔ-cytoplasmic DsRed larvae were scored for the number of contacts. Probability of a cell being Lz+ increases linearly with the number of cells it is in contact …
Figure 4—figure supplement 1
In HmlΔ-nuclearDsRed; Lz-GAL4, UAS-GFP larvae, the dorsal cluster in the A7 segment is easily observed (left panel).
After physical manipulation, the number of cells in the cluster is severely reduced (middle panel). 1 hr 30 min after manipulation clusters are re-established (right panel).
Figure 4—figure supplement 2
Flow cytometry analysis for cell viability and Dot-GAL4 lineage tracing in cluster disrupted larvae.
Disrupting the clusters does not increase the percentage of dying hemocytes and does not induce the release of hemocytes from the lymph gland. Numbers represent means ± SEM.
Figure 4—figure supplement 3
It is possible to detect lymph gland derived hemocytes by flow cytometry with Dot-GAL4 lineage-traced hemocytes in pupa (blue line) or when larvae are infected with parasitoid wasps (green line).
In contrast, lymph gland-derived hemocytes are virtually absent from L3 larvae in homeostasis (red line).
Videos
Video 1
Induction of lozenge expression in hemocyte clusters.
HmlΔ-nuclearDsRed; Lz>EGFP hemocytes in a dorsal cluster. Examples of Hml+Lz− hemocytes in division are highlighted with green circles and examples of Hml+Lz− hemocytes differentiating into Hml+Lz+ …
